Mechanistic dissection of novel regulators of TDP-43 aggregation identified in a genome-wide CRISPR-Cas9 knockout screen

全基因组 CRISPR-Cas9 敲除筛选中发现的 TDP-43 聚集新型调节因子的机制剖析

基本信息

批准号：
10268168
负责人：
Katelyn Sweeney
金额：
$ 3.35万
依托单位：
UNIVERSITY OF PENNSYLVANIA
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-06-01 至 2023-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10268168
关键词：
ALS patients Address Affect Alzheimer&apos s Disease Amyotrophic Lateral Sclerosis Antiviral Agents Astrocytes Binding Biology CRISPR/Cas technology Cell Line Cells Cellular Morphology Client Coculture Techniques Collaborations Coupled Cyclic AMP-Dependent Protein Kinases Data Development Diffuse Disease Disease Progression Dissection Double-Stranded RNA Enzyme-Linked Immunosorbent Assay Fluorescence-Activated Cell Sorting Generations Genes Genetic Predisposition to Disease Global Change Goals Homeostasis Immune Immune response Immune signaling Importins In Vitro Incidence Interferon-alpha Knock-out Link Mass Spectrum Analysis Measures Mediating Messenger RNA Methyltransferase Microglia Mind Modeling Modification Molecular Chaperones Motor Neurons Mutation Neurodegenerative Disorders Nuclear Nuclear Export Pathogenesis Pathologic Pathology Pathway interactions Phase Physiologic pulse Prevalence Proteins Quality Control RNA RNA Binding RNA methylation RNA-Binding Proteins Regulation Reporter Ribosomal RNA Role Signal Pathway Signal Transduction Small RNA Stains Structure Testing Therapeutic Time Toxic effect Translations Western Blotting Work Yeast Model System Yeasts base crosslinking and immunoprecipitation sequencing disease-causing mutation follow-up frontotemporal degeneration genome-wide in vitro testing interest novel prevent protein TDP-43 protein kinase R shape analysis therapeutically effective

项目摘要

Project Summary The mislocalization and aggregation of RNA-binding proteins (RBP) is a pathological hallmark of Amyotrophic Lateral Sclerosis (ALS). The RBP TAR DNA-binding protein 43 (TDP-43) is of particular interest, as pathological aggregation of TDP-43 is observed in nearly 97% of ALS patients, despite the fact that disease-causing mutations in TDP-43 explain fewer than 5% of disease incidence. This observation suggests that misregulation or mutation of other genes converge on TDP-43 pathology, however it remains poorly understood how the aggregates arise or whether they can be reversed or prevented. To this end, we have developed Pulse-Shape Analysis-based aggregation reporters for the TDP-43 that, when coupled to fluorescence activated cell sorting, quantifies aggregation at the single cell level. This reporter was leveraged against a genome-wide CRISPR- Cas9 knockout screen to identify regulators of TDP-43 aggregation. Reassuringly, this screen revealed known interactors and pathways involved in TDP-43 regulation and pathology. Additionally, this work revealed several novel proteins not previously implicated in TDP-43 biology or aggregation. Initial follow up on one top hit, SRRD, revealed that this protein reduces TDP-43 aggregation in a mammalian aggregation model, and reduces TDP- 43-associated toxicity in a yeast model, indicating that these genes indeed modify TDP-43 aggregation. The goal of this proposal is to mechanistically dissect how these novel regulators modify TDP-43 aggregation. Using an induced motor neuron model of TDP-43 aggregation, yeast toxicity models, and in vitro studies, I will assess the interactors, signaling pathways, and mode of TDP-43 modification for the following four genes: METTL5, EIF2AK2, XPO4, and SRRD. This work will not only reveal the function of uncharacterized proteins, but dissect their heretofore unknown mechanisms in regulating TDP-43 aggregation. Through better understanding of how TDP-43 aggregation is regulated, the field can begin to answer questions regarding the role of TDP-43 aggregation in disease pathogenesis and progression. By understanding the regulatory network of underlying TDP-43 aggregation, we can begin to therapeutically modulate the pathways that contribute to disease.

项目概要 RNA 结合蛋白 (RBP) 的错误定位和聚集是肌萎缩侧索硬化症的病理标志侧索硬化症（ALS）。 RBP TAR DNA 结合蛋白 43 (TDP-43) 特别令人感兴趣，因为它具有病理学特征。在近 97% 的 ALS 患者中观察到 TDP-43 聚集，尽管事实上 TDP-43 突变只能解释不到 5% 的疾病发病率。这一观察表明，监管不当或其他基因的突变与 TDP-43 病理学一致，但目前仍知之甚少。聚集的出现或是否可以逆转或预防。为此，我们开发了 Pulse-Shape TDP-43 基于分析的聚合报告器，当与荧光激活细胞分选结合时，量化单细胞水平的聚集。该记者利用全基因组 CRISPR- Cas9 敲除筛选，以确定 TDP-43 聚集的调节因子。令人放心的是，这个屏幕显示了已知的参与 TDP-43 调节和病理学的相互作用因子和途径。此外，这项工作还揭示了一些以前未涉及 TDP-43 生物学或聚集的新蛋白质。对热门歌曲 SRRD 的初步跟进，研究表明，该蛋白可减少哺乳动物聚集模型中的 TDP-43 聚集，并减少 TDP- 酵母模型中与 43 相关的毒性，表明这些基因确实改变了 TDP-43 聚集。该提案的目标是从机制上剖析这些新颖的监管机构如何修改 TDP-43 聚合。使用 TDP-43 聚集的诱导运动神经元模型、酵母毒性模型和体外研究中，我将评估以下四个方面的相互作用因子、信号通路和 TDP-43 修饰模式基因：METTL5、EIF2AK2、XPO4 和 SRRD。这项工作不仅揭示了未表征的功能蛋白质，但剖析了它们迄今为止未知的调节 TDP-43 聚集的机制。通过更好了解了 TDP-43 聚合是如何监管的，该领域可以开始回答有关 TDP-43 聚合的问题 TDP-43 聚集在疾病发病机制和进展中的作用。通过了解底层 TDP-43 聚合的监管网络，我们可以开始治疗性调节导致疾病的途径。